Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 9, 2012 to May 21, 2013
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Health Effects guidelines, OPPTS 870.3550, Reproduction/ Developmental Toxicity Screening Test. EPA 712-C-00-367, July 2000.
Commission Regulation (EC) No. 440/2008, L 142, Annex Part B, May 30, 2008
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München , Germany
Limit test:
no

Test material

Constituent 1
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name: FAT 40825/B TE
Batch No.: BOP 01-12
Physical State: powder
Colour: Black
Storage Conditions: at room temperature
pH: 6 to 8
Density: 1.688 g/cm3 at 19.5 ºC
Purity: 84.8% all coloured organic constituents, main constituent: 63.7%
Date of Analysis: 18 July 2012
Expiry Date: 28 January 2017
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test System:
Species/strain: Healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: males: 10-11 weeks old, females: 10-11 weeks old.

Body weight at the allocation of the animals to the experimental groups:
males: 264 - 314 g (mean: 295.53 g, ±20% = 236.42 – 354.63 g)
females: 183 - 217 g (mean: 199.89 g, ±20% = 159.91 – 239.86 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.

Housing and Feeding Conditions

- Full barrier in an air-conditioned room
- Temperature: 22 ±3 °C
- Relative humidity: 55 ±10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0702)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept individually in IVC cages (except during the mating period when one female will be paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 190612)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
water
Details on exposure:
The test item was weighed into a tarred plastic vial on a suitable precision balance and the vehicle (sterile water) was added to give the appropriate final concentration of the test item.

The vehicle has been selected as suggested by the sponsor and on the basis of the test item’s characteristics. The test item formulation was prepared freshly on each administration day before the administration procedure. The time of preparation was recorded for all dosing formulations.

Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration.
The vehicle was also used as control item.

The followig doses were evaluated
Control: 0 mg/kg Body weight
Low Dose: 100 mg/kg Body weight
Mid Dose: 300 mg/kg Body Weight
High Dose: 1000 mg/kg Body Weight

The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received aqua ad injectionem (sterile water) using the same dose volume.

Dose volumes were adjusted individually based on the weekly body weight measurements. The administration volume was 5 mL/kg body weight.

Details on mating procedure:
Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the pregnancy. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation. The cages were arranged in such a way that possible effects due to cage placement were minimised.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Each dosing concentration was analysed with respect to the target nominal concentration. Stability and homogeneity of the test item in the vehicle were analysed for the low and high dose concentrations.
Samples for the nominal concentration verification were taken in study week 1 (first week of pre-mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation) from all groups (16 samples).
Samples for homogeneity analysis were taken from the top, middle and bottom of the high dose and the low dose formulation in study week 1 and 5 (12 samples).
Samples for stability analysis were taken in the first week of the study, 0 hours after the preparation and another sample 6 hours after the preparation (at room temperature), from high and low dose formulations (4 samples).
All samples of dosing formulations were sent to Analytic department of BSL BIOSERVICE Scientific Laboratories GmbH on particular day of sampling.
Formulation analysis were performed in accordance with GLP and the procedures followed for the determination of test item concentration in the dosing formulations and control formulation was described in a separate study phase plan (BSL phase study No. 122441) issued by the Principal Investigator which was amended to study plan.
Duration of treatment / exposure:
The female animals were treated with the test item formulation or vehicle on 7 days per week basis for a period of 54 days, i.e. during 14 days of pre-mating and 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females.
Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.
Frequency of treatment:
7 days/week
Details on study schedule:
Date of Draft Study Plan: 25 May 2012
Date of Final Study Plan: 01 June 2012
Date of 1st amendment 13 June 2012
to study plan

Date of Start of Experiment 31 May 2012
Study Initiation Date: 17 September 2012
1st Amendment to Study Plan: 14 December 2012
Experimental Starting Date: 26 September 2012
Experimental Completion Date: 19 November 2012
Completion Date of
Delegated Phase (Histopathology): 14 February 2013

Completion Date of
Delegated Phase
(Formulation Analysis): 06 March 2013

Date of Draft Report (BSL): 20 March 2013
Date of Final Report (BSL): 21 May 2013

Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg body weight
Basis:
nominal conc.
No. of animals per sex per dose:
80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group). This included the control group animals shared with BSL study No.122430.
Control animals:
yes, concurrent vehicle
Positive control:
Not included

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
Time Schedule: Daily
General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

BODY WEIGHT:
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study.
During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups. Any animals prematurely sacrificed were weighed prior to the sacrifice.

FOOD CONSUMPTION:
Food consumption was measured weekly on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.
Oestrous cyclicity (parental animals):
Not evaluated
Sperm parameters (parental animals):
Not Evaluated
Litter observations:
The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.

Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by writing numbers on the back with the help of a permanent marker or by tattooing. In addition to the observations of parent animals, any abnormal behaviour of the offspring was recorded.
Postmortem examinations (parental animals):
SACRIFICE:
The males were sacrificed any time after the completion of the mating period (minimum total dosing of 28 days). Females were sacrificed on respective post-partum day 4 along with the pups by using an anaesthesia (ketamine/xylazin, 2:1, medistar Arzneimittel, lot no: 00212, expiry date: 03/2014 and Serumwerk, lot no: 00711, expiry date: 08/2013) was used.

Females not delivered were sacrificed on day 26 after the sperm-positive vaginal smear as an evidence of mating.

GROSS NECROPSY:
All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. The number of corpora lutea and implantation sites was recorded for any females sacrificed 24 to 26 days after the end of the pairing period with no evidence of mating and for any females sacrificed on day 25 post-coitum due to non-delivery.

Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), and all organs showing macroscopic lesions of all adult animals were preserved in 10 % neutral buffered formalin, except for eyes, testes and epididymides which were preserved in modified Davidson’s Solution and then transferred in 10 % neutral buffered formalin.

HISTOPATHOLOGY/ORGAN WEIGHTS
The testes and epididymides of all male adult animals as well as the ovaries, uterus with cervix of all female adult animals were weighed.
Paired organs were weighed separately.

A full histopathological evaluation (after the preparation of paraffin sections and haematoxylin-eosin staining) of testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) from C and HD group animals and all organs showing gross lesions except discolorations due to the color of the test item were examined. Histopathological examinations of testis and epididymis were also extended to animals of the intermediate dose groups.
A detailed qualitative examination of the testes was made taking into account the tubular stages of the spermatogenic cycle at the evaluation of additional haematoxylin-PAS (Periodic Acid Schiff) stained slides.
The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory Propath UK Ltd, Willow Court, Netherwood Road, Hereford HR2 6JU, Great Britain (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory KALEIDIS – Consultancy in Histopathology (test site for histopathology), 6 rue du Gers, 68300 Saint-Louis, France. Blocking, embedding, cutting, H&E staining and scientific slide evaluation was performed according to the corresponding SOP’s of the test sites.

Postmortem examinations (offspring):
Pups sacrificed on day 4 post partum were carefully examined externally for gross abnormalities.
Statistics:
A statistical assessment of the results of the body weight, food consumption, litter data and absolute and relative organ weights was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.5.01 software (p<0.05 is considered as statistically significant).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

Mortality:
No mortalities were observed at any dose levels of male and female animals.

Clinical Observations:
No test item related clinical signs were observed in male and female animals during the entire study period. However, few specific findings observed in few male and female animals of MD and HD group were slight to moderate piloerection, moving the bedding, alopecia on various body parts and slight to moderate salivation. As these clinical signs were observed on few days of the treatment period and in absence of any effect on parameters of general health like body weight and food consumption in treatment groups, these findings were considered to be non adverse.

Body Weight Development:
In male and females, no statistically significant effect on body weight and body weight gain was observed throughout the study period in treatment groups and all group mean values for body weight and body weight gain were comparable with respective controls.

Food Consumption:
In males and females, statistical analysis of food consumption data revealed no statistically significant effect on food consumption except significant decrease during gestation day 7-14 in HD group females when compared with controls. As no test item related effect on body weight development was observed during that period, this significant effect on food consumption was considered to be non adverse. In correlation to the body weight and body weight gain, the food consumption in both males and females increased with the progress of the study.

Pathology:
At necropsy of male (after minimum total dosing of 28 days - on day 29) and females (on post-natal day 4) by using a high dose of Ketamine/Xylazine (2:1), macroscopic examination of the animals revealed no test item related macroscopic findings in males and females. Few spontaneous gross pathological findings observed in male and female animals were yellow spots on epididymides, dark discolouration of various organs like testes, seminal vesicles with coagulating gland, prostate, kidneys, mesenteric lymph nodes, ovaries, uterus, oviduct and cervix. These findings related to discolouration in male and females were attributed to the colour of the test item and as such not a systemic effect due to the test item administration.

Organ Weight:
In males and females, no statistically significant effect on absolute and relative (to body weight) organ weights was observed in any treatment group when compared with the controls.

Histopathology:
At necropsy, dark discoloration of a number of organs, comprising mainly reproductive organs, kidney and lymph nodes, was observed in all males and in the majority of females treated at 1000 mg/kg/day. At 300 mg/kg/day, testis or kidney was dark discoloured in a proportion of males. These colour changes were considered to be caused by the colour of the test item and were not evaluated histologically in tissues other than reproductive organs.

Histologically, brown pigment in interstitial macrophages was seen at a minor degree in the testis and epididymis in males treated at 1000 mg/kg/day. At 300 mg/kg/day, brown pigment in epididymal macrophages was seen in one single male, only. These changes were considered to represent deposition of the test item or its degradation product(s) in macrophages and, in the absence of other structural changes or indication of functional impairment of the organs concerned, were considered non-adverse.

No test item-related histological findings were noted in the other male and in the female reproductive organs. One control female and one female treated at 1000 mg/kg/day did not show any indication of recent pregnancy at terminal sacrifice. This was not considered test item-related.
As a conclusion, test item-related histopathological findings noted in this study were considered to represent test item deposition in macrophages and to be non-adverse. Based on the extent of histopathological evaluation as defined by the study plan, the NOAEL (No Observed Adverse Effect Level) for pathology was therefore considered to be 1000 mg/kg/day under the conditions of this study.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Only gross external observations were made
Histopathological findings:
not examined

Details on results (F1)

Litter Data:
No treatment related effect was observed on total number of pups born, number of males, number of females, sex ratio, live pups, still birth and runt on PND 0 and number of males, number of females, total number of pups and sex ratio on PND 4.
All group mean and individual values for various litter data parameters from treatment groups were comparable with the controls.

Litter Weight Data:
Statistical analysis of litter weight data revealed no treatment related effect on group mean litter weight, total litter weight, male litter weight and female litter weight on PND 0 and PND 4 when compared with corresponding controls.

Precoital interval and duration of gestation:
No treatment related effect was observed on precoital interval and duration of gestation and values were comparable between the groups. All pregnancies resulted in normal births.
Successful mating resulted in 9, 10, 10 and 9 pregnancies in the control, low, mid and high dose respectively.

Pre and Postnatal Data:
Pre and post natal data like group mean number of corpora lutea, number of implantation sites, number of pups born on PND 0, percent preimplantation and post implantation loss remained unaffected due to treatment when compared with controls.

Reproductive Indices:
No treatment related effect on copulation index, delivery index, fertility index and viability index was observed when compared with controls.
Reduced fertility index (No. of pregnant females/No. of copulated females X 100) was observed in C and HD (90 %) dose group as compared to LD and MD groups (100 %).

Pup Survival Data
No significant effect on survival of the pups from PND 0 to PND 4 was observed in any treatment group when compared with controls.
Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treatment groups and no pup mortality was observed in this study except one pup from LD group female 59 (pup No. 10) was found dead on PND 4. One pup from HD group female 72 (pup No. 1) went missing on PND 2 and was presumed to be cannibalized by the dam. Since this incidence of cannibalism was observed in one female and it was within the rat cannibalism rate, this incidence was not considered to be test item related.

Pup External Findings:
No treatment related gross external findings were observed in pups from any of the treated groups on PND 0 and 4. However, following few external findings were observed:
In control females, one pup from female 45 (pup No. 3) was observed for hematoma at head on PND 0.
In MD females, one pup from female 63 (pup No. 5) was observed for dark snout on PND 0.
In HD females, one pup from female 72 (pup No. 1) was small (runt).
All these findings were considered to be spontaneous in nature and not related to the treatment with test item.



Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, the repeated dose administration of FAT 40825/B TE in sterile water to the male (28 days) and female (maximum 54 days) Wistar rats at dosages of 100, 300 and 1000 mg/kg body weight revealed neither mortalities nor findings of toxicological relevance regards reproductive performance or developmental effects.

Based on the data generated from this reproduction/ developmental toxicity screening test with FAT 40825/B TE, the no observed adverse effect level (NOAEL) is considered to be 1000 mg/kg body weight for reproduction/ developmental toxicity screening in males and females.

Executive summary:

The aim of this study was to assess the possible effects of FAT 40821/A on male and female fertility and embryofetal development after repeated dose administration in Wistar rats.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 54 days, i.e. during 14 days of pre-mating and 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. Animals of an additional control group were handled identically as the dose groups but received aqua ad injectionem (sterile water), the vehicle used in this study. The 4 groups comprised 10 male and 10 femaleWistar rats.

During the period of administration, the animals were observed each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.

After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum.

The males were sacrificed after completion of the mating period on treatment day 30 and the females along with their pups were sacrificed on post natal day 4. Non-pregnant females were sacrificed on day 26.

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Pups sacrificed on postnatal day 4 and those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation (after the preparation of paraffin sections and haematoxylin-eosin staining) of testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) from C and HD group animals and all organs showing gross lesions except discolorations due to the color of the test item were examined. Histopathological examinations of testis and epididymis were also extended to animals of the intermediate dose groups.


 The following doses were evaluated:

Control:                         0        mg/kg body weight

Low Dose:                    100     mg/kg body weight

Medium Dose:              300     mg/kg body weight

High Dose:                    1000   mg/kg body weight

The test item formulation was prepared freshly on each day of administration. The test item was dissolved in aqua ad injectionem (sterile water) and administered daily during 14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 3 in females. Males were dosed for 28 days. Dose volumes were adjusted individually based on weekly body weight measurements. Theadministration volume was 5 mL/kg body weight.

  

Summary Results

Mortality

No mortality occurred in the control or any of the dose groups during the treatment period of this study.

Clinical Signs

No test item related clinical signs were observed in male and female animals during the entire study period. However, few specific findings observed in few male and female animals of MD and HD group were slight to moderate piloerection, moving the bedding, alopecia on various body parts and slight to moderate salivation

Body Weight Development

In male and females, no statistically significant effect on body weight and body weight gain was observed throughout the study period in treatment groupsand all group mean values for body weight and body weight gain were comparable with respective controls.

Food Consumption

In males and females, statistical analysis of food consumption data revealed no statistically significant effect on food consumption except significant decrease during gestation day 7-14 in HD group females when compared with controls

Litter Data

No treatment related effect was observed on total number of pups born, number of males, number of females, sex ratio, live pups, still birth and runt on PND 0 and number of males, number of females, total number of pups and sex ratio on PND 4.

Litter Weight Data

Statistical analysis of litter weight data revealed no treatment related effect on group mean litter weight, total litter weight, male litter weight and female litter weight on PND 0 and PND 4 when compared with corresponding controls.

Precoital Interval and duration of Gestation

No treatment related effect was observed on precoital interval and duration of gestation and valueswere comparable between the groups. All pregnancies resulted in normal births.

Pre and Post Natal Data

Pre and post natal data like group mean number of corpora lutea, number of implantation sites, number of pups born on PND 0, percent preimplantation and post implantation loss remained unaffected due to treatment when compared with controls.

Reproductive Indices

No treatment related effect on copulation index, delivery index, fertility index and viability index was observed when compared with controls.

Reduced fertility index (No. of pregnant females/No. of copulated females X 100) was observed in C and HD (90 %) dose group as compared to LD and MD groups (100 %).

Pup Survival Data

Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treatment groups and no pup mortality was observed in this study except one pup from LD group female 59 (pup No. 10) was found dead on PND 4. One pup from HD group female 72 (pup No. 1) went missing on PND 2 and was presumed to be cannibalized by the dam.

Pup external Findings

No treatment related gross external findings were observed in pups from any of the treated groups on PND 0 and 4. However, few spontaneous findings like hematoma at head in one pup from control group female and dark snout in one pup from MD group females was observed on PND 0.

All these findings were considered to be spontaneous in nature and not related to the treatment with test item.

Pathology

At necropsy of male and females, macroscopic examination of the animals revealed no test item related macroscopic findings in males and females. Few spontaneous gross pathological findings observed in male and female animals were yellow spots on epididymides, dark discolouration of various organs like testes, seminal vesicles with coagulating gland, prostate, kidneys, mesenteric lymph nodes, ovaries, uterus, oviduct and cervix.

Organ Weight

In males and females, no statistically significant effect on absolute and relative (to body weight) organ weights was observed in any treatment group when compared with the controls.

Histopathology

Histologically, brown pigment in interstitial macrophages was seen at a minor degree in the testis and epididymis in males treated at 1000 mg/kg/day. At 300 mg/kg/day, brown pigment in epididymal macrophages was seen in one single male, only. These changes were considered to represent deposition of the test item or its degradation product(s) in macrophages and, in the absence of other structural changes or indication of functional impairment of the organs concerned, were considered non-adverse.

No test item-related histological findings were noted in the other male and in the female reproductive organs. One control female and one female treated at 1000 mg/kg/day did not show any indication of recent pregnancy at terminal sacrifice. This was not considered test item-related.

As a conclusion, test item-related histopathological findings noted in this study were considered to represent test item deposition in macrophages and to be non-adverse. Based on the extent of histopathological evaluation as defined by the study plan, the NOAEL (No Observed Adverse Effect Level) for pathology was therefore considered to be 1000 mg/kg/day under the conditions of this study.

Dose Formulation Analysis

Concentration analysis of formulation samples was determined in study week 1, 3, 5 and 7 for all dose groups. The mean recoveries observed in LD, MD and HD groups were 97.2%, 102.4% and 95.6% of the nominal concentration, respectively.

Stability of formulation samples was investigated in study week 1 for LD and HD dose groups. After 6 hours storage at -20 °C recovery compared to starting value was 96.2% and 101.3%.

Homogeneity of formulation samples was determined in study week 1 and 5 for LD and HD dose groups. The mean recovery observed for LD dose group was 111.8 and 96.6% of the nominal value, and 92.7 and 102.8% for HD dose group. The coefficients of variation of the different sampling locations (top, middle, bottom) were 1.6 and 7.0% in LD dose group, and 0.7 and 2.7% in HD dose group. 

Conclusion:    

In conclusion, the repeated dose administration ofFAT 40825/B TE in sterile water to the male (28 days) and female (maximum 54 days) Wistar rats at dosages of 100, 300 and 1000 mg/kg body weight revealed neither mortalities nor findings of toxicological relevance.

Based on the data generated from this reproduction/developmental toxicity screening test with FAT 40825/B TE, the no observed adverse effect level (NOAEL) is considered to be 1000 mg/kg body weight for reproduction/developmental toxicity screening in males and females.